Method of determining dTk isoenzyme activity and the use thereof

ABSTRACT

A method of determining dTk isoenzyme levels of a human or animal body fluid or cell sample is disclosed, which comprises the steps of reacting said sample with a substrate for said isoenzyme in the presence of a phosphate donor and a buffer system, and measuring the amount of phosphorylated product formed, said amount being proportional to said isoenzyme level. The method is characterized by using in combination 
     (a) a buffer system at pH ranging from 5 to 9 and being present in a concentration of no more than 250 mM, 
     (b) a substrate consisting of 2&#39;-deoxy-5-halouridin, part of which has a radio-labelled 5-halogen, and being present in a concentration ranging from 2×10 -9  -5×10 -6  M, and 
     (c) the phosphate donor being present in a concentration not exceeding 20 mM. 
     The sensitivity of the method permits measurement of minute amounts of dTk, and the method can be used for e.g. diagnosis, prognostics and monitoring of diseases involving alterated ATP mediated dTk activity, such as cancers, tumors, and certain virus infections, or alterated CTP mediated dTk activity, such as HSV type 1, type 2, and VZV infections.

The present invention relates to a method of determining the levels ofthe isoenzyme dTk (deoxythymidine kinase) in human or animal body fluidsor cell samples. The invention further relates to the use of said methodfor the diagnosis and prognostics of diseases dominated by ATP(adenosinetriphosphate) mediated dTk activity, such as cancers, tumoursand certain viral infections as well as of diseases characterized by CTP(cytidinetriphosphate) mediated dTk activity, such as HSV (HerpesSimplex Virus) type 1 and type 2 and VZV (Varicells Zoster Virus)infections. The invention also relates to the use of said method for dTkisoenzyme typing.

BACKGROUND OF THE INVENTION

The enzyme deoxythymidine kinase (dTk) provides the eucaryotic cell witha means for utilizing deoxythymidine (dT), which is not an intermediatein the thymidylate synthesis de novo. For this reason dTk is considereda salvage enzyme, introducing dT into the DNA metabolism. As the majordTk form of mammalian cell is only present during cell division, the dTkhas been denominated scavenger enzyme.

Three different cellular isoenzymes in human cells have been described.The cytosolar dTk, called dTk-F, which occurs in optimal amounts individing cells (stages Gl to S) (Bello, Exptl. Cell Res. 89: 263, 1974;Littlefield, Biochim. Biophys. Acta. 115: 398, 1966) and is more or lessabsent in resting cells. In humans this enzyme is coded for inchromosome 17 near the galactokinase locus. The second cellularisoenzyme is the mitochondrial, denominated dTk-A, which is present inthe mitochondrial matrix. The activity of this dTk remains relativelyconstant during the different cell stages (Adelstein et al; Develop.Biol. 216: 537, 1971), and dTk-A is coded for by chromosome 16. Thethird dTk, a minor activity called dTk-B, has only been reported incontinuous cell lines HeLa and KB and is said to be confined to theinside of the mitochondrial membrane (reviewed by Kit, Pharmacol. Ther.4: 501, 1979).

The three cellular dTks differ in biochemical properties. The dTk-F anddTk-B, which are quite similar, are distinguished, besides as tolocalization, by isoelectric focusing, having different pI, and byelectrophoretic mobility. In contrast to dTk-F and dTk-B, the dTk-Aaccepts cytidinetriphosphate (CTP) as a phosphate donor and is not assensitive as the others to dTTP (deoxythymidinetriphosphate) feedbackinhibition. The dTk-A also phosphorylates deoxycytidine (dC) and isinhibited by dCTP (reviewed by Kit, Pharmacol. Ther. (4: 501, 1979).

With regard to viruses, specific isoenzymes, coded by the viral genome,have been shown in the cell after infection with viruses from the Herpesgroup and the Pox group. Enzymatically the human virus specific dTksresemble dTk-A, except for the vaccinia dTk which cannot utilize CTP asphosphate donor and not either can phosphorylate deoxycytidine. This dTkis easily distinguished from the human cellular dTks by electrophoresis(Kit et al., Progr. Med. Virol. 21: 13, Karger Basel 1975). Both the HSVdTks and the VZV dTk have a broader spectrum of possible phosphatedonors and accept different pyrimidines and pyrimidine analogues assubstrates (Cheng et al, Biochim. Biophys. Acta. 452: 370, 1976, and J.Virol. 31: 172, 1979). The competitive blocking of dTk isoenzymemediated dT conversion to dTmp (deoxythymidinemonophosphate) exerted bythe dT-analogue 2'-deoxy-5-iodourine (IUdR), has been known for a longtime. The use of radio-labeled IUdR directly as a substrate to gain highsensitivity in assays of viral dTks was shown by us (Gronowitz &Kallander, Infec. Immun. 29: 425, 1980).

As mentioned dTk-F occurrence in cells is coupled to cell proliferation,and it is more or less absent in the differentiated cell (Munch-Petersen& Tyrsted, Biochim. Biophys. 478: 364, 1977). Studies of dTk activity intransplantable mouse tumours have revealed high dTk activities withcorrelation to growth rate (Bresnick et al, Cancer Res. 29: 1969, andCancer Res. 31: 743, 1971). Recent reports have demonstrated enhanceddTk-F in peripheral blood lymphocytes of some patients suffering frommalignant non-Hodkin's lymphoma and lymphatic leukemia (Ellims et al,Cancer Res. 41: 691 and Brit. J. Haematol. 49: 479, 1981). Theseresearchers have also shown enhanced serum dTk levels in some patientsfrom the non-Hodkin's group (Ellims et al, Blood 58: 926, 1981). Due tothe conventional dTk assay used, employing ³ H-dT in high concentration(5×10⁻⁶ M), dTk activity could only be found in a few patients withadvanced disease. Further the dTk activity could be evaluated as aprognostic marker only if differential analysis regarding dTk-F anddTk-A was performed.

Conventionally dTk activity has thus been measured by following theconversion of either ³ H- or ¹⁴ C-labeled dT to dTmp with ATP as thephosphate donor. We have recently designed an improved dTk assay systemfor viral dTk isoenzymes, which uses ¹²⁵ I-Iododeoxyuridine (IUdR) asthe substrate. The improved sensitivity obtained allowed the detectionof dTk from as little as 25 HSV-infected cells. In contrast theconventional assay based on the use of ³ H-dT, at a normally usedconcentration of 10⁻⁵ M, would require at least 450 times more enzyme.However, even though being useful in studies of viral dTks this improvedassay system was not appropriate for long time assays of cellular dTk-F,partly due to the instability of this enzyme. The inability of thismethod of detecting the presence of minute dTk amounts in serum iseasily extracted from the studies over occurrence of virus dTk blockingantibodies (dTk-ab). In these studies more than 275 human sera wereassayed for dTk-ab but disturbing serum dTk activities were found inonly two cases (Gronowitz & Kallander, Infec. Immun. 29: 425, 1980;Gronowitz & Kallander, J. Med. Virol. 8: 177, 1981; Kallander et al,Infec. Immun. 36: 30, 1982).

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a means for thediagnosis of certain virus infections and malignant tumours, includingthe monitoring of the malignancies for different purposes.

A further object of the invention is to provide a sensitive assay systemcapable of detecting minute amounts of dTk present in human and animalbody fluids and cell samples, e.g. in serum, blister secretes, spinalfluid, vesicle fluid, and similar clinical specimens, which system isalso designed to minimize the contribution of cellular dTk-A.

A further object of the invention is to provide a method of assaying fordTk-F for use in the detection and prognostics of tumour diseases, andfor the monitoring of alterations in the disease, and detection ofrelapses.

A still further object of the invention is the use of said assay methodin the evaluation of metastases in connection with lung cancer of theoat cell type.

Still another object of the invention is the use of said assay method inconnection with leukemia for determining, by measurement of dTk presentin spinal fluid, if metastases are present in the cerebro-spinal system.

Another object of the invention is the use of said assay method as agroup specific marker for certain infections in connection with viraldiseases.

A further object of the invention is the use of a modification of saidassay method for achieving specific detection of herpes virus specificdTks present in e.g. serum or vesicle fluid.

The above and other objects of the invention will be explained in detailin the following description of the assay method and its applicationsaccording to the invention.

The invention thus provides an improved assay system for the measurementof dTk levels, especially dTk-F levels in e.g. serum. This assay systemis considerably more sensitive than prior art systems, and it has beenfound that the sensitivity is so high, that it is possible to use theassay system, which is very simple to practice, not only for determiningpathological dTk levels, but even normal levels in healthy individuals(compare FIGS. 1 and 6). This assay system was not only found to be verysensive and reproducible for dTk-F determinations, giving linearturnovers for more than two hours, but was also found to give 2-6 timeshigher turnovers than prior art systems with the viral dTks. Anotherimportant advantage, compared to the conventional assay system, is thecomparatively low turnover of dTk-A, this isoenzyme giving minimalcontribution when measuring dTk-F or viral dTk in non-purified samples.

A variant of this assay system, using CTP instead of ATP as thephosphate donor, was found to be useful for specific detection of herpesvirus induced dTk when samples to be analysed were preincubated withanit-herpes viruses dTk blocking sera.

The advantageous and unexpected technical effects described herein areaccording to the invention obtained by the use of a combination of abuffer system of low concentration and ionic strength, a specific typeof radio-labelled substrate, which is present in low concentration, anda low concentration of a phosphate donor.

In its broadest aspect the invention relates to a method of determiningdTk isoenzyme levels in a human or animal body fluid or cell samplewherein said sample is reacted with a substrate for said isoenzyme inthe presence of a phosphate donor and a buffer system, and wherein theamount of phosporylated product formed is measured, said amount beingproportional to said isoenzyme level. According to the invention saidmethod is characterized by using, in combination.

(a) a buffer system at pH ranging from 5 to 9 and being present in aconcentration of no more than 250 mM,

(b) a substrate consisting of 2'-deoxy-5-halouridin, part of which has aradiolabelled 5-halogen, and being present in a concentration rangingfrom 2×10⁻⁹ -5×10⁻⁶ M, and

(c) the phosphate donor being present in a concentration not exceeding20 mM.

In contrast to the earlier described assay system using ¹²⁵ I-IUdR assubstrate, the assay system according to the invention gives a linearturnover of substrate for the cellular dTk-F for more than two hours,and the detection sensitivity was drastically increased, as can be seenin FIG. 1. This Figure is a comparison between the assay systemaccording to the invention (--o--o--o) and said prior art assay system(--Δ--Δ--Δ--) (Gronowitz and Kallander, Infec. Immun. 29: 425, 1980).A=two sera containing human dTk-F originating from patients withleukemia. B=HSV type 2 dTk of cell culture origin. Total available ¹²⁵I-IUdR was 550×10³ cpm.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and B are diagrams showing enzyme activity plotted against timeand illustrating the drastically improved sensitivity of the claimedmethod compared to the closest prior art.

FIGS. 2A and B illustrate--by way of tlc graphs--the reduction inradiochemical purity of the preferred substrate according to theinvention, ¹²⁵ I-IUdR, on storage.

FIG. 3 is a diagram illustrating the calculation of enzyme activity inunits when using an internal substrate control.

FIG. 4 is a diagram illustrating the inhibitory effeft of high serumconcentrations in dTk measurements.

FIG. 5 illustrates--by way of diagrams--the typing of viral dTk indifferent clinical specimens.

FIG. 6 is a diagram illustrating the normal distribution of dTk activityin blood donors and pregnant women.

FIG. 7 is a diagram illustrating dTk activities, measured according tothe invention, in sera from patients with infectious mononucleosis andrelated to time after onset of illness.

FIG. 8 is a diagram illustrating dTk activities found in sera frompatients suffering from various virus infections, mycoplasma pneumoniae,and psittacosis.

FIGS. 9a and 9b are diagrams illustrating the correlation between serumdTk activity, as measured according to the invention, and the stage ofthe disease for patients suffering from NHL.

FIGS. 10a and 10b illustrate the probability of survival for NHLpatients related to the pretreatment serum dTk levels as measuredaccording to the invention.

FIGS. 11 and 12 are diagrams illustrating longitudinal studies of serumdTk levels for NHL patients with varying clinical course.

DESCRIPTION OF PREFERRED EMBODIMENTS

In a preferred embodiment of the invention said buffer system is presentin a concentration of less than about 150 mM, especially being about 100mM. The pH of the buffer is preferably in the range 6.5-8, especiallyabout 7.4. The best results have been obtained with buffers devoid ofmaleate, and lacking reactive or primary amino groups, in particularbuffers substantially devoid of TRIS-maleate, HEPES buffer being theespcially preferred buffer.

The substrate should be a radio-labelled thymidine analogue, especiallya 2'-deoxy-5-halogen-uridine, wherein the label preferably is a reactivehalogen isotop. The preferred substrate is ¹²⁵ I-2'-deoxy-5-iodouridine.The preferred substrate concentration is in the range of 1×10⁻⁸ -5×10⁻⁷M, especially about 10⁻⁷ M.

The preferred phosphate donor for dTk-F determination is ATP, and thepreferred phosphate donor for viral dTk determination is CTP. Thepreferred concentration of the phosphate donor is less than about 10 mM,especially about 0.5-5 mM. Phosphate donors known per se to have thesame isoenzyme specificity as ATP and CTP may also be used.

Mg²⁺ should preferably be present in at least equimolar concentrationrelative to the phosphate donor. A reducing agent, such asdithiothreitol, is preferably also present.

The measurement of the phosphorylated product can be made by proceduresknown per se (see e.g. Gronowitz et kallander, Infec. Immun. 29: 425,1980).

The application of the assay system according to the invention fordetection of dTk activities in clinical specimens, e.g. serum or blistersecrete, provided a new tool for diagnosis of certain virus infections,and malignancies. Also, normal serum dTk levels (e.g. >10 units),obtained with a single dTk assay with the procedure described, werefound to be an excellent marker for prognostic use and so alsosequential dTk values in monotoring the progression or regression ofmalignant disease. Furthermore in these patients serum dTk levels wereuseful as a marker of relapses and in evaluation of therapy effects.All, as examplified below.

In the preferred embodiment, which has been practiced when performingthe tests reported below, ¹²⁵ I-IUdR is used as the substrate. Thecomposition and proportions, including final concentrations, of thepreferred assay system are shown in Table 1 below.

                  TABLE 1    ______________________________________    Composition of buffer used in dTk assay    Component     Assay mixture                              Final concentration.sup.a    ______________________________________    .sup.b HEPES  0.1     M       86     mM    MgCl.sub.2    17      mM      15     mM    KCl           20      mM      17     mM    NaF           1,2     mM      1,0    mM    ATP           4.6     mM      3.9    mM    Dithiothreitol                  2.7     mM      2.3    mM    Bovine Albumine                  0.33    mg/ml   0.28   mg/ml    Glycerol      6.6%        5.7%    JUdR          --          1.1 × 10.sup.-7 M    pH            7.4         7.4    ______________________________________     .sup.a Proportions of one double sample is: 51.5 μl assay mixture, 6     μl of substrate solution (final concentration of .sup.125 IJUdR 1     × 10.sup.-7 M, 130-160 Ci/mM), and 2.5 μl of enzyme solution e.g     serum (added when starting the reaction), giving a final volume of 60     μl.     .sup.b N--2hydroxyethylpiperazine N--2ethanesulfonic acid.

In general, one double sample is prepared, consisting of 51.5 μlreaction solution, which is mixed with 6 μl substrate solutionimmediately before use. The reaction is then started by addition of 2.5μl enzyme solution (e.g. serum sample). From this standard volume of atotal of 60 μl, two samples of each 25 μl are taken. The composition ofassay mixtures for experiments consisting of several double samples,taken after different incubation times, are calculated by multiplyingthe volumes of the components in the standard solution with the desirednumber of double samples. The assay is performed at 37° C. and allcomponents are prewarmed 2 minutes before starting the reaction. Theenzyme reaction is terminated by pipetting the sample onto a 1 cm² pieceof Whatman DEAE-81 paper kept at 90°-100° C. To separate the productfrom the substrate, the paper is washed four times in 6 mMammoniumformate solution, once in distilled water and finally inmethanol. The washings are performed in 1 liter glass vessels kept onmagnetic stirring. 10-15 paper discs, kept in a colander, are processedsimultaneously. The colander is moved to a new washing bath every fiveminutes. Finally the paper discs are counted in an automatized gammacounter.

In order to eliminate day-to-day variation in the dTk assay, thereaction velocities obtained were recalculated to units. The reason forutilizing units in defining enzyme quantities is the variation ininitial radiochemical purity, combined with the found biological decayof commercially available ¹²⁵ I-IUdR, which makes total radioactivityadded per samle non-relevant for enzyme activity calculations. See FIG.2, which shows separation of batches of commercially obtained ¹²⁵ I-IUdRon thin layer chromatography using precoated silica gel plates (Merck 60F₂₅₄). 15% methanol, 85% chloroform was used as eluent. A=profiles atday of delivery, indicating 62% radiochemical purity. B=the same batchafter 150 days in refrigerator. Initially >90% radiochemical purity wasclaimed by the manufacturers. Instead, an internal biologic control,measuring accessible radioactivity, was included in each assay. Thiscontrol was a 100-fold excess of an HSV type 2 dtK preparation, givingextensive substate exhaustion. The amount of radioactivity incorporatedin a 1 hour assay by this control was found to be about 85% of thevalues of intact .sup. 125 I-IUdR, as calculated from thin layerchromatography. Considering the level (85%) found for the boilogicalcontrol, 1 unit of enzyme will be the enzyme amount converting 4.3×10⁻¹⁵moles of substrate per hour (under the conditions described). Thisrelationship between unit and approximative molar turnover was chosen,as 1 unit will practically be expressed as about 1000 cpm, with theamount of isotope normally used (cf. Table 1). Another advantage withthis biological isotope control, as it is processed in the same manneras the test samples, is that possible variations in units due tovariations in product recovery are eliminated. The procedure for thecalculation of units is exemplified in FIG. 3. Values for both thesample (--o--o--) and the control (--Δ--Δ--) are shown. A is the valueof the internal control in cpm, which is proportional to the totalavailable radiolabeled substrate. B is the value of product formed bythe investigated sample expressed as cpm per hour. ##EQU1## where (S) isthe substrate concentration (M), and v is the sample volume (liters).

Determination of serum dTk activities

Quantification of serum dTk was done in microtiter plates. For every 20serum samples, background controls and an internal substrate control(see above) were included, and the set was subsequently processed as aunit. The amounts of serum sample used were 5 μl (or less per 120 μlfinal volume, as more serum might be inhibitory See FIG. 4. dTkactivities were determined by sampling at 120 minutes. Enzyme activityas plotted against serum concentration per 60 μl sample. (o--o) serumfrom an individual suffering from NHL, (Δ--Δ) serum from an individualsuffering from active CLL. This inhibitory effect is probably due to thepresence of nucleotides or nucleosides, such as e.g. thymidine,thymidinetriphosphate, or catabolic enzymes present in some sera. Doublesamples were taken after 60 and 120 minutes. Medium turn-over, per hourand 25 μl sample, after correction for background, was calculated fromeach double sample separately. The average velocity value of the 60 and120 minutes sampling was then determined, and used in furthercalculations (see above), if <20% variation was found.

Determination of dTk activities present in spinal fluid

Essentially the same procedure as for serum was used. The specificactivity of ¹²⁵ I-IUdR was increased to around 320 Ci/mM. Spinal fluidsmay be concentrated before the enzyme determinations.

Qualitative determination of dTk isoenzymes

Clincal specimens, e.g. vesicle fluids or serum, containing dTk activitywere preincubated with isoenzyme specific dTk blocking antibody andresidual enzyme activity was determined according to the proceduredescribed above. ATP in the assay mixture was replaced by CTP, resultingin exclusion of dTk-F activity. The specimens to be investigated wereserially twofold diluted (usually starting at 1:6) in reaction solution,and 15 μl was transferred from each dilution to four wells in a microtiter plate, in order to achieve four identical dilution sets.Prediluted VZV dTk-blocking serum (25 ml) was added to one well, a HSVtype 1 dTk-blocking serum to a second well, a HSV type 2 dTk-blockingserum to a third well and to the last well a negative serum. Themixtures were incubated at 37° C. for 90 min., allowing theenzyme-antibody reaction to occur. Then the residual enzyme activitieswere determined. The reaction was started by the addition of 20 μl of asolution composed of 6 μl substrate solution mixed with 14 μl reactionsolution. For each dilution the values obtained in the presence of thedifferent antisera were recalculated as the percentage of the valuesobtained with the negative serum. Examples of such residual enzymeactivities plotted against the log₂ dilution of specimens are shown inFIG. 5, wherein the residual activity after incubation with anti-VZV dTkserum (Δ--Δ), anti-HSV type 1 dTk (serum ( -- ), and anti-HSV type 2serum (o--o)in relation to control incubation with negative serum isplotted against different dilutions of the following samples: A=vesiclefluid from an individual suffering from an HSV type 2 infection;B=vesicle fluid from an individual suffering from an VZV infection;C=vesicle fluid from an individual suffering from HSV type 1 infection;D=serum from an individual suffering from VZV infection. From theinhibition patterns the dTk type can be easily established. (See alsoTable 2 below).

                                      TABLE 2    __________________________________________________________________________    Detection and typing of viral dTk activity in clinical specimens.             Enzyme  % Residual enzyme activity    Sample         Sample             activity.sup.a                     after incubation with sera                                   Immunological    type code             (cpm × 10.sup.-3)                     VZV  HSV 1                              HSV 2                                   type of dTk                                           Clinical diagnosis    __________________________________________________________________________    Vesicle         1   2.5 × 10.sup.3                     17   92  123  VZV     Herpes zoster    fluid         2   8.1 × 10.sup.2                      7   118 85   VZV     Herpes zoster         3   6.6 × 10.sup.2                      6   119 104  VZV     Varicellae         4   --      --   --  --    --     vesicles non viral orgin         5   7.6 × 10.sup.1                     99   89   5   HSV 2   Genital Herpes         6   5.9 × 10.sup.2                      9   127 109  VZV     Varicellae         7   8.8 × 10.sup.1                     101  70   8   HSV 2   Genital Herpes         8   1.2 × 10.sup.3                     11   124 104  VZV     Herpes zoster         9   2.3 × 10.sup.2                     107   4  95   HSV 1   Herpes stomatitis         10  1.2 × 10.sup.1                     16   89  118  VZV         11  2.8 × 10.sup.3                      5   87  91   VZV     Varicellae         12  6.5 × 10.sup.2                      7   109 116  VZV     Varicellae         13  1.5 × 10.sup.2                     10   92  89   VZV     Generalized Herpes zoster    serum         A   2.8 × 10.sup.1                     18            VZV     Varicellae    samples         B   1.2 × 10.sup.2                     18            VZV     Varicellae         C   1.6 × 10.sup.2                      8   96  85   VZV     Varicellae         D   3.4 × 10.sup.1                     12   114 85   VZV     Varicellae         E   4.0 × 10.sup.1                     16            VZV     Varicellae         F   9.6 × 10.sup.1                     11            VZV     Varicellae    control         dTkA             1.0 × 10.sup.2                     120  111 98    dTk from         VZV 8.5 × 10.sup.1                      7   106 112    cultures:         HSV 1             1.3 × 10.sup.2                     96   11  94         HSV 2             7.5 × 10.sup.1                     112  89    9    __________________________________________________________________________     .sup.a Activity of control (sample incubated with negative serum) when     using 5 μl sample and IUdR at a specific activity of 370 Cl/mmole.

One important feature of the invention is thus the drastically increaseddetection sensitivity, which allows measurement even of normal serumlevels of dTk-F in healthy individuals, as can be seen from FIG. 6,which shows the distribution of dTk activity found in sera from 99 blooddonors (unfilled columns) and 25 pregnant women in the first trimester(shaded columns). The average is 2.4 units/μl with a standard deviationof 1.25.

Investigations of serum samples from individuals suffering frominfectious diseases lead to the discovery of 10-40 times elevated dTklevels in serum from patients in acute stages of certain virusinfections, e.g. mononucleosis infectiosa, rubella- and morbillivirusinfections. FIG. 7 shows the dTk activity/μl formed in sera frompatients with infectious mononucleosis as related to time after onset ofillness. Symbols labeled with the same letter indicate sera from thesame patient, and the dotted line shows the normal value for healthyindividuals. FIG. 8 shows the dTk activity/μl found in acute () andconvalescent (o) sera of patients suffering from different virusinfections, mycoplasma pneumoniae, and psittacosis. The dotted lineindicates a value, which is 4 times the normal value for healthyindividuals.

Concerning herpes viruses the results indicate elevated serum dTkactivities in connection with primary infections. No consequentincreases in dTk activities (>10 units) were found in connection withother infections (FIG. 8). The characterization of the serum activitiesfound has hitherto demonstrated the presence of Varicella Zoster Virus(VZV) specific dTk (Table 2) and cell dTk-F. It has also beendemonstrated that the claimed method can be used for determining virusspecific dTk present in vesicle fluids for rapid diagnosis of infections(Table 2). All serum dTk activities found in connection with viralinfections disappeared gradually, reaching normal levels within 2-6weeks.

Regarding patients suffering from malignant diseases, a limited study,using the assay method according to the invention, showed elevated serumdTk levels not only in patients suffering from malignancies originatingfrom the lympho-proliferative system, but also in patients havingmalignancies of other cells with metastases in the lymphoproliferativesystem (see Table 3).

Extended studies have been performed with well defined large serummaterials from patients suffering of lympho-proliferative diseases, suchas non-Hodgkin's lymphoma (NHL), Hodgin's disease (HD), cronic andactivated lymphatic leukemia (CLL) and myelomas. Detailed results in theNHL group were as follows: normal serum dTk and enhanced dTk levels ofdifferent magnitude were found. These dTk levels correlated to stagingof the disease, (see FIG. 9a which shows serum-dTk in 155 untreatedpatients with NHL correlated to stage of the disease), i.e., the moreadvanced disease the higher the dTk values. When the dTk levels wererelated to the malignancy of the tumour cells (classification accordingto the Kiel system), then a good correlation between high dTk levels andmalignancy was found (see FIG. 9b which shows serum dTk in 101 NHLpatients in stages III-IV divided into three stages of malignancy)

                  TABLE 3    ______________________________________    dTk levels in sera from patients with different tumour diseases.                               dTk activity    Type of disease                   Status of disease                               (units/μl)    ______________________________________    Lung cancer.sup.d                   no metastases                               5    Lung cancer    no metastases                               2    Lung cancer    metastases  163    Lung cancer    metastases  19    HD             metastases  108    HD             metastases  28    HD             no metastases                               4    HD             no metastases                               3    NHL            advanced stage                               333    NHL            advanced stage                               14    NHL            early stage 3    NHL            early stage 5    Adenocarcinoma active      72    Leukemia       acute       40    Leukemia       acute       185    B-cell leukemia                   active      52    CML            active      3500.sup.a    CML            active      45    CLL            active      62    CLL            active      18    CLL            semiactive  11    CLL            semiactive  12    CLL            in active   2.8.sup.b    Healthy individuals        2.4.sup.c    Leukemia acute no metastase                               0.07.sup.e    Leukemia acute no metastase                               0.20.sup.e    Leukemia acute metastase   1.3.sup.e    Leukemia acute metastase   1.6.sup.e    ______________________________________     .sup.a Therapy two days earlier.     .sup.b Average value from 6 patients; values ranging between 0.5 and 5.4     .sup.c See previous section.     .sup.d All lung cancers in this study were of the oat cell type.     .sup.e determination of dTk activity in spinal fluid, 5 μl undiluted     spinal fluid was used per 120 μl final volume in assay.     Abbreviations used;     HD; Hodgkins' disease     NHL; nonHodgkin's lymphoma     CML; chronic myeloic leukemia     CLL; chronic lymphatic leukemia

The utility of the assay method according to the invention forprognostic purposes was demonstrated by highly significant differencesin survival times between patient groups constructed by divisionaccording to measured dTk values <10 units and >10 units respectively.FIG. 10a shows the probability of survival for NHL patients in stagesIII-IV with pretreatment serum dTk <10 units (--o--o--, n=50) and >10units (-- -- , n=51). The numbers in parenthesis indicate remainingindividuals at observation after 32 months. FIG. 10b shows theprobability of survival for NHL patients in stages III-IV with "highgrade" malignancy of tumour. Pretreatment level of serum dTk <10(--o--o, n=11) and >10 units (-- -- --, n=27). The numbers inparenthesis indicate remaining individuals at observation after 32months.

Furthermore, longitudinal studies of serum dTk levels in NHL patientsrevealed alterations correlating to alterations of the disease, i.e.lower values upon regression of disease, higher upon progression andunaltered in constant disease. FIG. 11 shows the variations in themeasured serum dTk levels for A three patients with progressive disease,B two patients treated to partial remission, and C four patients treatedto complete remission. In FIG. 12 serum dTk is followed longitudinallyin patients, where both progressive disease (a), and remission (b)occur. (A) were two patients with progressive disease, who after changeof therapy were treated to partial remission before the diseaseprogressed again. (B) were three patients treated to remission (A. H.and E. L. to partial, and G. E. to complete remission). All of themlater relapsed. A. H. responded for a short time once more to therapy.These results clearly show the possibility the effect of therapy, andalso to rapidly detect relapses both during and after treatment asincreasing dTk values.

When using the assay method according to the invention in connectionwith the other above mentioned lympho-proliferative diseases variableserum dTk levels were found, e.g. in cronic CLL normal values werefound, while half activated CLL gave low but pathological values (>10units), and active CLL high. Possible uses of dTk levels, as examplifiedfor NHL, are also relevant for CLL. Moreover, studies of spinal fluid,derived from patients with leukemia, show presence of dTk when leukemiccells are present in the cerebro-spinal system. Regarding cancer, theresults found from patients with lung cancer of the oat cell typeindicate the possibility to utilize serum dTk levels as a marker ofmetastases as well as for other cancers. These conclusions are evidentfrom Table 3.

Naturally one has to be aware of possible transient elevated serum dTklevel in patients with malignant disease, due to the above mentionedvirus infections. However, these virus diseases are normally contractedin childhood, while malignant tumour diseases commonly is present inadults. Another factor relevant for the use of serum dTk levels for thedescribed purposes, is the rare occurence of elevated dTk activitiesindicated in a study of an unselected serum material, derived frompatients with undefined disease, consecutively obtained for diagnosticpurposes. Studies of medical records for these and other patients haveshown that neither pathological values in liver function tests, norleucocytosis are normally accompanied by enhanced serum dTk levels.

We claim:
 1. A method of determining dTk isoenzyme levels of a human oranimal body fluid or cell sample, comprising the steps of reacting saidsample with a substrate for said isoenzyme consisting of2'-deoxy-5-halouridin, containing a radio-labelled 5-halogen, saidsubstrate being present in a concentration ranging from 2×10⁻⁹ to 5×10⁻⁶M, in the presence of a phosphate donor in a concentration sufficientfor producing a measurable amount of phosphorylated product, saidconcentration not exceeding 20 mM and a buffer system sufficient formaintaining a pH within the range of 5 to 9 in a concentration of nomore than 250 mM, and measuring the amount of phosphorylated productformed on said substrate, said amount being proportional to isoenzymelevel.
 2. A method according to claim 1, wherein said pH is from about6.5-8, said substrate concentration is from 10⁻⁸ -5×10⁻⁷ M, and saidphosphate donor concentration is from about 0.5-5 mM.
 3. A methodaccording to claim 1 or 2, wherein said buffer substance is essentiallyfree from TRIS-maleate.
 4. A method according to claim 1, wherein saidphosphate donor is selected from the group consisting of ATP and CTP. 5.A method according to claim 1, further comprising the step ofdetermining available radio-labelled substrate by reacting a controlconsisting of dTk isoenzyme in an excess sufficient to cause substrateexhaustion, said control being processed in the same manner as saidsample before measuring the radioactivity of said phosphorylatedproduct.
 6. The method of claim 1, wherein said buffer system comprisesN-2-hydroxyethylpiperazine N-2-ethanesulfonic acid.
 7. The method ofclaim 1, wherein said buffer system is devoid of reactive or primaryamino groups.
 8. The method of claim 1, wherein substrate is ¹²⁵1-2-deoxy-5-iodouridine and the concentration of said substrate rangesfrom 1×10⁻⁸ to 5×10⁻⁷ M.